For over twenty years it has been proposed that calcium acts as a secondary messenger during stimulus-secretion coupling. Only recently, however, has it been shown that the concentration of free cytosolic calcium ([Ca2+]i) of pancreatic acinar cells increases following stimulation by secretagogues. Using the calcium-selective fluorescent indicator quin-2, the PI has demonstrated that a rapid, five-fold enhancement of [Ca2+]i occurs in acinar cells following stimulation by either the hormone cholecystokinin or carbachol, an analog of the neurotransmitter acetylcholine. One goal of this proposal is to characterize the properties of this transient rise in [Ca2+]i and to investigate its role in enzyme secretion. These studies will be performed on quin-2 loaded acinar cells. Although calcium appears to play an important role during stimulus-secretion coupling, the source of the calcium used to elevate [Ca2+]i and the mechanism by which this calcium is released are unclear. The plasma membrane and the endoplasmic reticulum have been proposed as important calcium-storage locations. Studies described here will investigate this possibility, to include investigation of the mechanism(s) by which calcium is released from these stores. Highly purified membrane vesicle preparations of plasma membrane and endoplasmic reticulum will be prepared from pancreatic acinar cells. Calcium-transport and calcium-stimulated ATPase activity will be characterized in each membrane fraction. Changes occurring in these activities during stimulus-secretion coupling will be examined by the following approaches. First, the effect(s) of secretagogues added directly to the membrane vesicle preparation will be determined. Second, calcium-transport and calcium-stimulated ATPase activity of membranes prepared from cells stimulated by secretagogues will be characterized and compared to control. Finally, the effect(s) of water-soluble inositol phosphates on these activities will be investigated. These compounds have been suggested as the messenger linking secretagogue's plasma membrane receptors to intracellular calcium stores. In additional studies, single channel recordings will be made from membrane fragments inserted in lipid bilayers on patch-clamp pipettes. Any ion channels present will be characterized and the effects of secretagogues and/or water-soluble inositol phosphates on these channels will be determined. The studies described here will further clarify calcium's role in the stimulus-secretion coupling process of pancreatic acinar cells and the mechanism(s) controlling [Ca2+]i during thid process.